Conservation at the uterine-placental interface

Abstract

The hemochorial placentation site is characterized by a dynamic interplay between trophoblast cells and maternal cells. These cells cooperate to establish an interface required for nutrient delivery to promote fetal growth. In the human, trophoblast cells penetrate deep into the uterus. This is not a consistent feature of hemochorial placentation and has hindered the establishment of suitable animal models. The rat represents an intriguing model for investigating hemochorial placentation with deep trophoblast cell invasion. In this study, we used single-cell RNA sequencing to characterize the transcriptome of the invasive trophoblast cell lineage, as well as other cell populations within the rat uterine-placental interface during early (gestation day [gd] 15.5) and late (gd 19.5) stages of intrauterine trophoblast cell invasion. We identified a robust set of transcripts that define invasive trophoblast cells, as well as transcripts that distinguished endothelial, smooth muscle, natural killer, and macrophage cells. Invasive trophoblast, immune, and endothelial cell populations exhibited distinct spatial relationships within the uterine-placental interface. Furthermore, the maturation stage of invasive trophoblast cell development could be determined by assessing gestation stage-dependent changes in transcript expression. Finally, and most importantly, expression of a prominent subset of rat invasive trophoblast cell transcripts is conserved in the invasive extravillous trophoblast cell lineage of the human placenta. These findings provide foundational data to identify and interrogate key conserved regulatory mechanisms essential for the development and function of an important compartment within the hemochorial placentation site that is essential for a healthy pregnancy.

Keywords: placentation; rat; single cell genomic analysis; trophoblast.

Fig. 1.

Single cell interrogation of the rat uterine–placental interface. (A) Schematic showing isolation of the rat uterine–placental interface from gd 15.5 or 19.5. Uterine tissue at the site of trophoblast invasion was peeled away from placental tissue and associated decidua. (B and C) Visualization of cell clusters from gd 15.5 and 19.5, respectively, plotted using UMAP. (D and E) Dot plots for gd 15.5 and 19.5 showing marker transcripts used to identify cell clusters. Dot size represents the average percentage of cells expressing the transcript. Dot colors correspond to cell types. For transcript expression level, see SI Appendix, Fig. S2 and Dataset S1. Abbreviations: UPI, uterine–placental interface; JZ, junctional zone; LZ, labyrinth zone.

Fig. 2.

Distribution of transcripts enriched in invasive trophoblast cells at the rat UPI. (A) Dot plots for gd 15.5 (Left) and 19.5 (Right) showing expression levels of invasive trophoblast cell enriched transcripts. Dot size and color represents the average percentage of cells expressing the transcript and the average level of expression, respectively. (B) Schematic depicting a gd 19.5 placentation site and a section through the central region of a gd 19.5 placentation site. Distribution of invasive trophoblast cells was determined by in situ hybridization of Prl7b1 transcripts. (Scale bar: 1,000 μm.) (C) Localization of invasive trophoblast cell enriched transcripts [Nppb, Plac1 (LOC102550080), Fstl3, Igf2, Tfpi; red] within the gd 19.5 placentation site. Transcripts were detected using in situ hybridization and colocalized to the distribution of Prl7b1 (green). (Scale bars Left and Middle: 1,000 μm; Right: 100 μm.) For abbreviations, see Fig. 1 legend.

Fig. 3.

Distribution of NK cells and macrophages within the rat uterine–placental interface. (A) NK cell and invasive trophoblast cells were monitored in gd 13.5, 15.5, and 19.5 placentation sites using in situ hybridization for Prf1 (red) and Prl7b1 (green), respectively. (B) Macrophages and invasive trophoblast cells were monitored in gd 13.5, 15.5, and 19.5 placentation sites using in situ hybridization for Lyz2 (red) and Prl7b1 (green), respectively. (Scale bar: 1,000 μm.) Arrowheads indicate examples of the distribution of NK cells (Top, Prf1 positive) and macrophages (Bottom, Lyz2 positive).

Fig. 4.

Differential invasive trophoblast cell gene expression on gd 15.5 versus gd 19.5. (A) Volcano plot showing differentially expressed genes (DEGs) in gd 15.5 and 19.5 invasive trophoblast cell clusters. Top 10 most significantly changed genes are labeled. (B) Bar plots showing selected significantly enriched gene ontology terms for transcripts up-regulated at gd 15.5 (Top) and transcripts up-regulated at gd 19.5 (Bottom). For full list of enriched terms, see Dataset S2). (C and D) Differential expression of LOC171573 and Cecam9 transcripts within placentation sites at gd 15.5 and gd 19.5. Transcripts were detected using in situ hybridization and colocalized to the distribution of Prl7b1 (green). Yellow arrows indicate the intrauterine invasive cells expressing LOC171573 and Ceacam9 at gd 15.5. (Scale bar: 1,000 μm.).

Fig. 5.

Conserved transcript expression in rat invasive trophoblast cells and human EVT cells. (A) Bar plots showing that transcripts highly expressed in the invasive trophoblast cell clusters at gd 15.5 and 19.5 in rats share similar profiles to EVT cells in human placenta. Enrichment analyses were carried out using human placenta single cell data as references: Vento-Tormo et al. (29), Suryawanshi et al. (28), Xiang et al. (30), Castel et al. (31), and Liu et al. (27). A significant enrichment has an adjusted P value ≤0.05, fold change ≥1.5, and number of observed genes ≥5. Red, significant enrichment; gray, insignificant enrichment. (B) Bar plot showing the number of rat invasive trophoblast cell genes (y axis) expressed in different human EVT cell datasets. Seven EVT cell datasets were analyzed (Dataset S4) and the x axis shows the total number of EVT cell datasets in which the rat invasive trophoblast gene was either an EVT cell marker or expressed in EVT cells. (C) Dot plots showing GO enrichment for rat invasive trophoblast cell genes that are also expressed in human placenta. Dot size represents the number of observed genes related to a term; dot colors correspond to log₂(fold change) of the terms. Selected gene ontology terms are shown. For a full list of enriched terms, see SI Appendix, Fig. S9. Abbreviations: SCT, syncytiotrophoblast; VCT, villous cytotrophoblast; Endo, vascular endothelial cells (villi); CTB, cytotrophoblast; EVT (8 wk), EVT from villi at 8 wk of pregnancy; EVT (24 wk), EVT from decidua at 24 wk of pregnancy; CTB (8 wk), CTB from villi at 8 wk of pregnancy.